Electron-transfer (ET) processes are vital elements of energy transduction pathways in living cells. The broad goal of this research program is to define the factors that control the rates of these reactions. Research is focused on defining how protein structure regulates electron flow; how rates depend on the energetics of the redox sites; how the electronic structures of the redox sites affect the directionality and efficiency of electron transfer; and how rates depend on protein conformation and dynamics. Aim 1: When the electronic coupling between redox sites in a protein is strong, and polypeptide relaxation dynamics are slow, theory suggests that ET rates are independent of coupling strength and are limited by the polypeptide relaxation dynamics. The effects of medium dynamics will be probed with investigations of ET in strongly coupled Ru- and Os-modified proteins. Aim 2: The efficiency of the coupling between redox centers is determined by the three-dimensional structure of the intervening polypeptide. ET rates in Ru-modified proteins with similar backbone structures but different primary sequences will be examined in order to determine the importance of side-chain atoms in mediating long-range couplings. Aim 3: There is a rapidly growing list of proteins in which amino-acid radicals (e.g., tryptophan, tyrosine) are believed to play key enzymatic roles. In order to assess their roles in enzyme catalysis, a detailed understanding of the kinetics and mechanisms of Trp and Tyr redox reactions must be developed. The kinetics of radical formation and reduction in Re- and Ru-modified azurins will be investigated to elucidate the interplay between proton- and electron-transfer reactions. Aim 4: In order that ET not become rate-limiting in biological redox processes, millisecond or faster tunneling times are required. For low driving forces, this requirement sets the maximum donor-acceptor distance for biological tunneling reactions at approximately 20 A. Multiple tunneling steps are necessary to effect electron transport over longer distances. Ru- and Re-modified azurins with intervening redox-active groups (e.g., 3-nitrotyrosine) will be designed and prepared in order to test experimentally the efficiency of multistep tunneling in mediating long-range electron transport.